The ultimate goal of this work is to understand the molecular mechanisms by which calcium regulates the contraction of vertebrate skeletal (actin-linked regulation) and molluscan (myosin-linked regulation) muscle. Although the mechanism of myosin and acto-myosin ATP hydrolysis have been extensively studied by rapid kinetic methods there is relatively little information about the mechanism of the complete system in which calcium regulation is maintained. The detailed mechanism of rabbit skeletal muscle acto-myosin will be reinvestigated using actin filaments containing the calcium sensitive regulatory proteins troponin and tropomyosin and myosin-Sl and heavymeromyosin (proteolytic fragments of myosin) that contain the calcium binding light chains. The mechanism of scallop acto-myosin ATP hydrolysis will be determined with heavymeromyosin that maintains the calcium sensitivity. The rate or equilibrium constants of each of the steps of the reaction mechanisms will be measured by stopped-flow and quenched-flow methods, while systematically varying the calcium concentration to determined which steps of the mechanism are calcium sensitive. A detailed understanding of the molecular mechanism of calcium regulation of acto-myosin ATP hydrolysis is fundamental to understand the mechanism of calcium regulation of muscle contraction and the regulation of non-muscle contractile systems that involve actin and myosin.